Immunoreactive and immunotherapeutic molecules

ABSTRACT

The present invention relates generally to molecules such as peptides, polypeptides and proteins which interact immunologically with antibodies or T-cells in subjects having pre-clinical or clinical Insulin-Dependent Diabetes Mellitus (IDDM). These molecules are preferentially immunoreactive to T-cells in subjects having pre-clinical or clinical IDDM and are useful in the development of diagnostic, therapeutic and prophylactic agents for IDDM.

[0001] The present invention relates generally to molecules such aspeptides, polypeptides and proteins which interact immunologically withantibodies or T-cells in subjects having pre-clinical or clinicalInsulin-Dependent Diabetes Mellitus (IDDM). These molecules arepreferentially immunoreactive to T-cells in subjects having pre-clinicalor clinical IDDM and are useful in the development of diagnostic,therapeutic and prophylactic agents for IDDM.

[0002] Amino acid sequences are referred to herein by sequence identitynumbers (SEQ ID NOs) which are defined at the end of the specification.

[0003] Throughout this specification, unless the context requiresotherwise, the word “comprise”, or variations such as “comprises” or“comprising”, will be understood to imply the inclusion of a statedelement or integer or group of elements or integers, but not to theexclusion of any other element or integer or group of elements orintegers.

[0004] Insulin-Dependent Diabetes Mellitis is a serious diseaseresulting from the destruction of insulin-secreting β-cells, probablymediated by T cells that recognise β-cell autoantigens. A major antigenimplicated in T-cell mediated β-cell destruction characteristic of IDDMis glutamic acid decarboxylase (GAD), which occurs in two majorisoforms, GAD 65 and GAD 67. These two isoforms have approximately 65%similarity at the amino acid sequence level. Subjects with IDDM or athigh-risk of the disease show autoantibody and autoreactive T-cellresponses to GAD insulin or both autoantigens. In NOD mice, an animalmodel for spontaneous IDDM, GAD is a dominant and early target antigen(Tisch et al Nature 366:72-75, 1993).

[0005] Identification of the immunodominant epitope(s) of pathogenicautoantigens involved in β-cell autoimmunity could lead to improvedmethods of diagnosis as well as therapeutic strategies to prevent IDDM.

[0006] In work leading up to the present invention, the inventors soughtto identify immunodominant epitopes in GAD and proinsulin molecules inorder to improve upon current diagnostic procedures and to furtherdevelop therapeutic and prophylactic compositions and treatmentapproaches for IDDM.

[0007] In accordance with the present invention, peptides weresynthesised based on a thirteen amino acid region of high similaritybetween the sequences of human GAD 65 (amino acid residue numbers506-518) and human proinsulin (amino acid residue numbers 24-36), whichregion of similarity also extends to human GAD 67 and mouse proinsulinsand mouse GADs (FIG. 1). The immunoreactivity of these peptides isidentified in accordance with the present invention on the basis ofinteractivity of peripheral blood cells or T-cells obtained from theperipheral blood of subjects with pre-clinical or clinical IDDM, therebyforming the basis for a new range of diagnostic, therapeutic andprophylactic procedures for IDDM.

[0008] Accordingly, one aspect of the present invention provides arecombinant or synthetic peptide or chemical equivalents thereof of theformula:

X₁X₂X₃

[0009] Wherein:

[0010] X₁ and X₃ may be the same or different and each is an amino acidsequence comprising from 0 to 40 naturally or non-naturally occurringamino acid residues; X₂ is any amino acid sequence of from 10 to 100residues derived from, homologous to or contiguous within amino acids506 to 518 inclusive or derivatives thereof of human GAD65 and/or aminoacids 24 to 36 inclusive or derivatives thereof of human proinsulin; andwherein said peptide molecule is capable of reacting with T cells andmodifying T-cell function when incubated with cells from subjects havingpre-clinical or clinical Insulin-Dependent Diabetes Mellitus (IDDM).Preferred cells include but are not limited to peripheral bloodmononuclear cells (PBMCs), anticoagulated whole blood and tissue biopsycells.

[0011] Reference to a “peptide” includes reference to a polypeptide orprotein or parts thereof.

[0012] In a preferred embodiment X₂ comprises not less than about 10 andnot greater than about 50, amino acid residues, more preferably not lessthan about 10 and not greater than about 30 amino acid residues and evenmore preferably not less than about 10 and not greater than about 15.

[0013] In a particularly preferred embodiment X₂ has either of thefollowing amino acid sequences: F F Y T P K T R R E A E D [SEQ ID NO:1];or F W Y I P P S L R T L E D [SEQ ID NO:23].

[0014] According to this preferred embodiment, there is provided arecombinant or synthetic peptide or chemical equivalent thereofcomprising the sequence:

X ₁ X ₂ X

[0015] Wherein

[0016] X₁ and X₂ may be the same or different and each is an amino acidsequence comprising from 0 to 15 naturally or non-naturally occurringamino acid residues; X₂ is selected from FFYTPKTRREAED and FWYIPPSLRTLEDor a derivative or chemical equivalent thereof and wherein said peptideis capable of reacting with T cells and modifying T-cell function whenincubated with cells from subjects with pre-clinical or clinical IDDMand determining reactivity by an appropriate assay. Preferred cellsinclude but are not limited PBMCs, anti-coagulated whole blood or tissuebiopsy cells and determining reactivity by an appropriate assay.

[0017] The peptides of the present invention may be prepared byrecombinant or chemically synthetic means. According to a preferredaspect of the present invention, there is provided a recombinant peptidewhich is preferentially immunologically reactive with

[0018] T-cells from individuals with clinical or pre-clinical IDDM,which is prepared by the expression of a host cell transformed with acassette coding for the peptide sequences of the present invention. Thepeptide may be fused to another peptide, polypeptide or protein.Alternatively, the peptide may be prepared by chemical synthetictechniques, such as by the Merrifield solid-phase synthesis procedure.The synthetic or recombinant peptide may or may not retain GAD activityor proinsulin activity. Furthermore, although synthetic peptides of theformula given above represent a preferred embodiment, the presentinvention also extends to biologically pure preparations of thenaturally occurring peptides or fragments thereof. By “biologicallypure” is meant a preparation comprising at least about 60%, preferablyat least about 70%, more preferably at least about 80% and still morepreferably at least about 90% or greater as determined by weight,activity or other suitable means.

[0019] By “pre-clinical IDDM” as used herein means those subjects whomay or may not be first degree relatives of someone with IDDM who havegenetic and/or immune markers of pancreatic islet (β) cell autoimmunity.By “immune markers” is meant amongst other parameters known to those inthe art to include circulating antibodies and/or T-cells reactive withislet (β) cell autoantigens.

[0020] By “derivatives” as used herein is taken to include any single ormultiple amino acid substitution, deletion and/or addition relative tothe naturally occurring amino acid sequence in the native molecule fromwhich the peptide is derived including any single or multiplesubstitution, deletion and/or addition of other molecules associatedwith the peptide, including carbohydrate, lipid and/or otherproteinacious moieties. Such derivatives, therefore, includeglycosylated or non-glycosylated forms or molecules with alteredglycosylation patterns.

[0021] By the term “reacting with T cells and modifying T-cell function”as used herein is taken to include T-cell activation, T-cellinactivation and/or T-cell death.

[0022] The present invention also covers chemical analogues of thesubject peptides which include, but is not limited to, modifications toside chains, incorporation of unnatural amino acids and/or theirderivatives, during peptide synthesis and the use of cross-linkers andother methods which impose conformational constraints on the peptides ortheir analogues.

[0023] Examples of side chain modifications contemplated by the presentinvention include modifications of amino groups such as by reductivealkylation by reaction with an aldehyde followed by reduction withNaBH₄; amidination with methylacetimidate; acylation with aceticanhydride; carbamoylation of amino groups with cyanate;trinitrobenzylation of amino groups with 2,4,6-trinitrobenzene sulphonicacid (TNBS); acylation of amino groups with succinic anhydride andtetrahydrophthalic anhydride; and pyridoxylation of lysine withpyridoxal-5′-phosphate followed by reduction with NaBH₄.

[0024] The guanidine group of arginine residues may be modified by theformation of heterocyclic condensation products with reagents such as2,3-butanedione, phenylglyoxal and glyoxal.

[0025] The carboxyl group may be modified by carbodiimide activation viaO-acylisourea formation followed by subsequent derivitisation, forexample, to a corresponding amide.

[0026] Sulphydryl groups may be modified by methods such ascarboxymethylation with iodoacetic acid or iodoacetamide; performic acidoxidation to cysteic acid; formation of mixed disulphides with otherthiol compounds; reaction with maleimide, maleic anhydride or othersubstituted maleimide; formation of mercurial derivatives using4-chloromercuribenzoate, 4-chloromercuriphenylsulphonic acid,phenylmercury chloride, 2-chloromercuri-4-nitrophenol and othermercurials; carbarnoylation with cyanate at alkaline pH.

[0027] Tryptophan residues may be modified by, for example, oxidationwith N-bromosuccinimide or alkylation of the indole ring with2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides. Tyrosine residueson the other hand, may be altered by nitration with tetranitromethane toform a 3-nitrotyrosine derivative.

[0028] Modification of the imidazole ring of a histidine residue may beaccomplished by alkylation with iodoacetic acid derivatives orN-carbethoxylation with diethylpyrocarbonate.

[0029] Examples of incorporating unnatural amino acids and derivativesduring peptide synthesis include, but are not limited to, use ofnorleucine, 4-amino butyric acid, 4-amino-3-hydroxy-5-phenylpentanoicacid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine,ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid,2-thienyl alanine and/or D-isomers of amino acids.

[0030] Crosslinkers can be used, for example, to stabilise 3Dconformations, using homo-bifunctional crosslinkers such as thebifunctional imido esters having (CH2)_(n) spacer groups with n=1 ton=6, glutaraldehyde, N-hydroxysuccinimide esters and hetero-bifunctionalreagents which usually contain an amino-reactive moiety such asN-hydroxysuccinimide and another group specific-reactive moiety such asmaleimido or dithio moiety (SH) or carbodiimide (COOH). In addition,peptides can be conformationally constrained by, for example,incorporation of C_(α) and N_(α)-methylamino acids, introduction ofdouble bonds between C_(α) and C_(β) atoms of amino acids and theformation of cyclic peptides or analogues by introducing covalent bondssuch as forming an amide bond between the N and C termini, between twoside chains or between a side chain and the N or C terminus.

[0031] The invention also extends to use of the peptides, or derivativesthereof of the present invention in the treatment of patients. In thislatter aspect, such methods of treatment include their use as anadsorbent to remove autoantibodies or autoreactive cells from a patient,their use in direct administration to a patient as a means ofdesensitising or inducing immunological tolerance or other mechanisms toeliminate or diminish reactivity of autoreactive T-cells orautoantibodies to IDDM autoantigens or to generate T-cell lines orclones to be used for or as therapeutic agents.

[0032] According to this aspect of the present invention, there isprovided a method of treatment comprising administering to a subject aneffective amount of a peptide or chemical equivalent thereof for a timeand under conditions sufficient to remove or substantially reduce thepresence or function in said subject of autoreactive T-cells and/orautoantibodies to IDDM autoantigens wherein the peptide comprises theformula:

X₁X₂X₃

[0033] Wherein:

[0034] X₁ and X₃ may be the same or different and each is an amino acidsequence comprising from 0 to 40 naturally or non-naturally occurringamino acid residues; X₂ is any amino acid sequence of from 10 to 100residues derived from, homologous, to or contiguous within amino acids506 to 518 inclusive or derivatives thereof of human GAD65 and/or aminoacids 24 to 36 inclusive or derivatives thereof of human proinsulin; andwherein said peptide molecule is capable of reacting with T cells andmodifying T-cell function when incubated with cells from subjects havingclinical or pre-clinical Insulin-Dependent Diabetes Mellitus (IDDM).Preferred cells include but are not limited to peripheral bloodmononuclear cells (PBMCs), anticoagulated whole blood and tissue biopsycells.

[0035] The method of treatment contemplated herein includes, but is notlimited to, the following examples. A first example of treatment isdesensitisation or tolerance induction using an effective amount ofsynthetic peptide or derivative thereof to alter T-cell recognition ofor response to GAD and/or pro-insulin and/or other IDDM antigens and/orinduce T-cell suppression or regulation. This may be achieved by usingthe known effect of certain ultraviolet wavelengths, especially UV-B, tomodify antigen presentation through the skin or transmucosal or systemicadministration. Effective amounts of the peptides or derivatives thereofwould be applied epicutaneously to the skin of subjects exhibitingperipheral blood T-cell reactivity to GAD or proinsulin peptides orpolypeptides. After exposure of skin to UV-B radiation, treatment wouldbe repeated until such time that T-cell reactivity to GAD or proinsulinwas suppressed.

[0036] A second example of treatment is to induce mucosal-mediatedtolerance using an effective amount of the subject peptides orderivatives thereof to alter T-cell recognition of or response to GADand/or pro-insulin and/or other IDDM antigens and/or induce T-cellsuppression using an effective amount of peptide or derivative thereofto alter T-cell recognition of or response to GAD and/or pro-insulinand/or other IDDM antigens and/or induce T-cell suppression by theadministration of the peptide or derivatives thereof by oral, aerosol orintranasal means amongst other routes of mucosal administration.

[0037] Another treatment involves application of the subject peptides tothe skin together with one or more cytokines such as but not limited toTNFα or β. A further treatment involves systemic administration ofsoluble peptide via subcutaneous or intravenous routes to induceimmunological tolerance. Yet another treatment involves T-cellimmunisation whereby T-cell lines are generated to GAD or proinsulinpeptide or polypeptide or fragments thereof by standard procedures,cells attenuated by fixation with agents such as glutaraldehyde orparaformaldehyde, washed under sterile conditions and re-injected intopatients for a time and under conditions to cause suppression of theendogenous T-cell response to autoantigens. These approaches areapplicable to the prevention of IDDM progression in asymptomaticsubjects with pre-clinical IDDM or subjects with recent—onset clinicalIDDM, as well as to the recurrence of IDDM in subjects who have receivedpancreas, islet cell or insulin-producing cell transplants. Theseapproaches are also applicable to Stiff Man Syndrome (SMS) and otherdiseases where GAD and/or proinsulin is an autoantigen.

[0038] In accordance with the present invention, the effective amount ofpeptide is 0.1 μg to 10 mg per dose and preferably 1.0 μg to 1 mg perdose. A dose may comprise a single administration or protocol comprisingsingle or multiple administration hourly, daily, weekly or monthly or atother suitable times. Administration may be by any convenient means suchas, but not limited to, intravenous, subcutaneous, epicutaneous,infusion, oral, topical, intranansal, aerosol suppository orintraperitoneal administration. The peptide may be administered alone orin combination with one or more other active molecules such as moleculeswhich facilitate the activity or action of the peptide for examplelipopolysaccharide (LPS), choleratoxin β-chain, Lymphocyte FunctionalAssociated Antigen-3 (LFA-3), other adjuvants and in particular, tumournecrosis factor α (TNF-α), tumour necrosis factor β (TNF-β) or leukaemiainhibitory factor (LIF).

[0039] In yet a further embodiment, the present invention contemplatesthe use of the peptides described herein to measure reactivity of asubject's cells to the IDDM autoantigen. The peptides or derivativesthereof may be added in solution or bound to a solid support togetherwith cells derived from peripheral blood or from tissue biopsies eitherunfractionated, fractionated or derived as continuous cell lines.Reactivity to the autoantigen may then be measured by standardproliferation assays such as incorporation of tritiated thymidine,standard cytotoxic assays such as release of marker radioactivity fromtarget cells, measurements of expressed or secreted molecules such assurface markers, cytokines or other standard assays of cellularreactivity which are well known in the art.

[0040] According to this aspect of the present invention, there isprovided a method of assaying the reactivity of a subject to IDDMautoantigen, said method comprising contacting a peptide or chemicalequivalent thereof comprising the formula:

X₁X₂X₃

[0041] Wherein:

[0042] X₁ and X₃ may be the same or different and each is an amino acidsequence comprising from 0 to 40 naturally or non-naturally occurringamino acid residues; X₂ is any amino acid sequence of from 10 to 100residues derived from, homologous to or contiguous within amino acids506 to 518 inclusive or derivatives thereof of human GAD65 and/or aminoacids 24 to 36 inclusive or derivatives thereof of human proinsulin; andwherein said peptide molecule is capable of reacting with T cells andmodifying T-cell function when incubated with cells from subjects havingpre-clinical or clinical Insulin-Dependent Diabetes Mellitus (IDDM) anddetermining reactivity by appropriate assay. In accordance with thisassay, any cell type may be used but is preferably selected from PBMC's,anti-coagulated whole blood cells or tissue biopsy cells.

[0043] Preferably, the present invention contemplates a method ofassaying the reactivity of a subject to IDDM autoantigen said methodcomprising contacting a peptide or chemical equivalent thereofcomprising the formula:

X₁X₂X₃

[0044] Wherein:

[0045] X₁ and X₂ may be the same or different and each is an amino acidsequence comprising from 0 to 15 naturally or non-naturally occurringamino acid residues; X₂ is selected from FFYTPKTRREAED and FWYIPPSLRTLEDor a derivative or chemical equivalent thereof and wherein said peptideis capable of reacting with T cells and modifying T-cell function whenincubated with cells from subjects with pre-clinical or clinical IDDMand determining reactivity by an appropriate assay. Preferably, cellsinclude but are not limited to peripheral blood mononuclear cells(PBMCs), anticoagulated whole blood and tissue biopsy cells.

[0046] In another embodiment of the present invention, there is provideda diagnostic kit for assaying T cells. Standard 96-well plates, as usedin ELISA, are pre-coated with a monoclonal antibody (MAb) to a T-cellcytokine such as γ-interferon (γ-IFN) with or without antigen.Alternatively, antigen is added in soluble form together with aliquotsof peripheral blood, peripheral blood mononuclear cells or T-cells.Incubation is allowed to proceed for one or more days, the supernatant(comprising medium and plasma) and the cells are washed off, wellswashed again and plates developed with a labelled second MAb to thecytokine such as anti-γ-IFN conjugated with alkaline phosphatase orhorseradish peroxidase. Colorimetric reaction and read-out systems canthen be utilised. Alternatively, soluble cytokines (eg: γ-IFN) aremeasured in the supernatant by standard assays such as ELISA; further itis possible to visualise microscopically by the ELISPOT techniqueindividual spots on bottoms of wells representing cytokine produced atthe single cell level thereby enabling the frequency ofpeptide-epitope-reactive T-cells to be determined.

[0047] The present invention will now be further described withreference to the following non-limiting Figures and Examples.

[0048] In the Figures

[0049]FIG. 1 shows a comparison of the regions of similarity among mouseand human proinsulins and GADs. Similarities are boxed; identitieswithin boxes are shaded. The C-terminus of the mature insulin B-chainand the pro-insulin cleavage site are indicated by the vertical line andarrow respectively.

[0050]FIG. 2 is a graphical representation showing the level of cellularproliferation expressed as the delta score following the stimulation ofperipheral blood mononuclear cells taken from IDDM at-risk (as describedin Example 1) or control subjects with the following peptides: humanGAD65 (residues 506-518); human proinsulin (residues 24-36); irrelevantcontrol peptide; or tetanus toxoid (CSL Ltd., Melbourne, Australia).

[0051]FIG. 3 is a graphical representation showing proliferation(mean+sem) of pbmc to proinsulin (aa 24-36) and insulin (aa 1-15) inpre-clinical and control subjects.

[0052]FIG. 4 is a graphical representation showing IFN-gamma response(mean+sem) to proinsulin (aa 24-36) and insulin beta chain (aa 1-15) inpre-clinical and control subjects.

[0053]FIG. 5 is a graphical representation showing IL10 response(mean+sem) to proinsulin (aa 24-36) and insulin beta-chain (aa 1-15) inpre-clinical and control subjects.

[0054] The following single and three letter abbreviations are used foramino acid residues: Three-letter One-letter Amino Acid AbbreviationSymbol Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic acid Asp DCysteine Cys C Glutamine Gln Q Glutamic acid Glu E Glycine Gly GHistidine His H Isoleucine Ile I Leucine Leu L Lysine Lys K MethionineMet M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr TTryptophan Trp W Tyrosine Tyr Y Valine Val V Any residue Xaa X

EXAMPLE 1 Subjects

[0055] Subjects at-risk for IDDM were from the Melbourne PrediabetesFamily Study, Victoria, Australia. Each was entered on the basis ofhaving at least one first degree relative with IDDM and islet cellantibodies (ICA)≧20 JDF units and/or insulin autoantibodies (IAA)≧100nU/ml. All had normal fasting blood glucose and glycated hemoglobin andhad had repeat antibody and metabolic tests at six monthly intervals.

[0056] Control subjects were HLA-DR matched, asymptomatic, and withouthistory of IDDM.

[0057] All subjects gave informed, signed consent and the study wasapproved by the Ethics Committees of the Royal Melbourne Hospital andthe Walter and Eliza Hall Institute of Medical Research. Details ofSubjects are described in Table 1.

EXAMPLE 2 HLA Typing and Assays of ICA, IAA, GAD Ab, FPIR

[0058] HLA Typing

[0059] HLA class I (A, B, C) and HLA class II (DR,DQ) typing wasperformed using populations of T and B lymphocytes respectively. Thecells were isolated from anticoagulated blood using magnetic beads(Dynal) coated with monoclonal antibodies to CD8 (class I) or amonomorphic determinant on the class II beta chain (class II). Theenriched cell populations were typed in a standardmicrolymphocytotoxicity assay using a battery of 240 allosera for classI and 120 allosera for class II.

[0060] Antibody Assays

[0061] ICA were assayed using indirect immunofluorescence on blood groupO donor pancreas. Titres, in JDF units, were determined by doublingdilution of positive sera and comparison with standard sera run in eachassay. The assay has been included in all International DiabetesWorkshops and proficiency programs.

[0062] IAA were assayed by a radiobinding assay which has beeninternationally standardised. The upper limit for normal control sera is40 nU insulin bound/ml serum.

[0063] GAD antibodies were assayed by immunoprecipitation of GADenzymatic activity from piglet brain extract. The mean plus (three) 3 SDof 72 healthy subjects, 460 nU/ml, was used to define the normal range.

[0064] First phase insulin release (FPIR)

[0065] FPIR was calculated as the sum of serum insulin concentrations at1 and 3 minutes following the completion of intravenous glucose (0.5g/kg body weight) injected over 3 minutes.

EXAMPLE 3 T-Cell Proliferation Assay

[0066] Blood was drawn from paired IDDM at-risk and HLA-DR matchedcontrols at the same time (within 30 minutes) and processed similarly toreduce the effects of diurnal variation and handling artefacts.Peripheral blood mononuclear cells were isolated from heparinised wholeblood by Ficoll-Paque (Pharmacia Biotech) density centrifugation, washedand resuspended in RPMI 1640 medium (Biosciences Pty Ltd) containing 20mM Hepes (CSL Ltd), 10⁻⁵ M 2-mercaptoethanol (BDR), penicillin 100U/ml), streptomycin (100 μg/ml) and 10% v/v autologous plasma. Aliquotsof 200 μl (2×10⁵ cells) were transferred into wells of a 96-well,round-bottomed plate (Falcon) and incubated in replicates of six withthe following peptides at final concentrations of 10, 2, and 0.4 μg/ml:human GAD65 (506-518), human proinsulin (24-36) (synthesised using anApplied Biosystems Model 431A synthesiser (ABI Foster City, Calif.), andan irrelevant control peptide (CRFDPQFALTNIAVRK) (MacromolecularResources, Fort Collins, Colo.). Tetanus toxoid (CSL Ltd, Melbourne,Australia) at final concentrations of 1.8, 0.18 and 0.018 LfU/ml wasused as a positive control. Twelve “autologous only” wells containingcells but without antigen were included as the background control.Plates were incubated at 37° C. in a 5% v/v CO₂ humidified incubator for6 days; 0.25 μCi of [³H]thymidine (ICN) was added to each well for thelast 6 hours. The cells were than harvested onto glass fibre filters andincorporated radioactivity measured by beta-particle counting (PackardModel 2000 Liquid Scintillation Counter). The level of cellularproliferation was expressed as the delta score (DS=mean counts perminute (cpm) incorporated in the presence of antigen, minus the mean cpmof the “autologous only” wells).

EXAMPLE 4 T-cell Proliferative Responses

[0067] T-cell proliferative responses to the similar 13-mer peptidesfrom proinsulin and GAD were compared for ten pairs of HLA-DR matchedat-risk and control subjects. HLA-DR matching was thought to beimportant not only because of the specificity of peptide binding to MHCclass II alleles but also because of the known association between MHCclass II and IDDM. Therefore, T-cell responses would reflect IDDM ratherthan MHC specificity . Responses to the highest concentration of eitherpeptide were significantly (proinsulin, p<0.008; GAD, p<0.018-Wilcoxonone-tailed paired analysis) greater among IDDM at-risk than controlsubjects. The results are summarised in Table 2.

[0068] Reactivity to the proinsulin sequence was confined almostentirely to IDDM at-risk subjects, whereas some controls also respondedto the GAD peptide (Table 2, FIG. 2) Both groups responded similarly totetanus, and no subject reacted to the unrelated control peptide.

[0069] For six of these pairs (#1, 2, 3, 5, 6, 7) the assay wasperformed on a separate occasion, but using twice as many cells (4×10⁵per well). Exhaustion of the media resulted in unreliable results inthree cases. In two of the other three (#5 and 6), the results wereconsistent with those tabulated here, while in the third (#3) theat-risk subject displayed greater reactivity to both antigens at thehigher cell number.

EXAMPLE 5 T-Cell Cytokine Secretion Assays

[0070] In a second cohort of 18 paired IDDM at-risk and HLA-DR-matchedcontrols, PBMCs indicated as per Example 3 were incubated with humanproinsulin 24-36and human insulin B chain 1-15 each at 0.5, 5 and 50μg/ml under the conditions as per Example 3. In addition to harvestingcells for the measurement of proliferation by ([³H] thymidine uptakeafter 6 days, as per Example 3, incubation media above the cells wassampled after 2 days for the measurement of IFN-γ and interleukin-(IL-)10 by standard ELISA methods.

EXAMPLE 6 T-Cell Responses

[0071] T-cell proliferative and IFN-γ and IL-10 secretory responses tohuman proinsulin 24-36 and human insulin B 1-15 were compared for 18pairs of HLA-DR matched IDDM at-risk and control subjects. As perExample 4, there was a significantly greater (p=0.003) proliferativeresponse of IDDM at-risk subjects to the proinsulin peptide (FIG. 3). Inaddition, both IFN-γ and IL-10 secretion in response to the proinsulinpeptide were significantly increased (p=0.005 and p=0.001, respectively)compared to matched control subjects (FIGS. 4, 5).

[0072] Those skilled in the art will appreciate that the inventiondescribed herein is susceptible to variations and modifications otherthan those specifically described. It is to be understood that theinvention includes all such variations and modifications. The inventionalso includes all of the steps, features, compositions and compoundsreferred to or indicated in this specification, individually orcollectively, and any and all combinations of any two or more of saidsteps or features. TABLE 1 HLA Subject # Age Years Follow-up A B DR DQICA * IAA † 1 14 1.6 1 8 3 2 160,69,56 4,30,−20 2 23 4.8 2 44,55 3,4 5,855,37,14,6,5,5 −25,9,41,−2,0,44 3 22 6.8 2,28 7,8 3,4 2,837,37,37,37,52,30,58,46,26 8,9,2,31,7,9,−41,−1,64 4 13 1.3 1,11 8,27 3,42,8 160,190 84,280 5 25 5.5 2 44,62 4,11 7,8 0,19,18,16,22,0,045,31,42,60,29,130,30 6 20 5.5 1,2 8,62 3,4 2,8 19.19.104.86.8480,560,400,130,300 7 18 1.7 1,3 8,18 3 2 69,69 13,20 8 9 3.2 1,2 8,443,4 2,8 160,160,160,160 −2,−26,36,59 9 10 2.8 1,2 8,27 3,4 2,8160,160,120,24 2,29,14,120 10 14 4.8 1,32 8,14 4,7 2,8 14,13,51,18240,490,470,1000 Subject # Age Years Follow-up GAD Ab £ FPIR ¶ 1 14 1.6118,155 2 23 4.8 278,602 124,113,57 3 22 6.8 1637,2259,634,1535183,155,140,161,56 4 13 1.3 79,91 5 25 5.5 736,936,1336,790,810137,143,68,15 6 20 5.5 937,2258,2389 105,238,165,128 7 18 1.7 44,47 8 93.2 2300,1830 118,129,87 9 10 2.8 1525,1388 26,56,29 10 14 4.8 432318,181,165

[0073] TABLE 2 Delta Scores* Proinsulin Proinsulin Proinsulin Autologous10 μg/ml 2 μg/ml 0.4 μg/ml Pair # At Risk Control At Risk Control AtRisk Control At Risk Control 1 881 2979 1391 0 459 0 1040 0 2 236 389351 0 0 0 33 0 3 6515 217 0 −64 355 43 0 0 4 595 1347 104 0 0 0 288 0 51745 1269 694 0 0 0 0 0 6 1007 265 397 98 65 380 0 0 7 1392 454 467 93 00 0 0 8 9993 308 2128 0 1367 0 0 0 9 598 135 0 0 265 13 0 0 10 597 87056 21 0 22 0 0 Mean 2355.8 823.4 558.7 27.6 251.1 45.8 136.0 0 Std.Error 1025.7 276.2 219.5 13.0 135.3 37.4 104.4 0 Wilcoxon P-Value 0.0080.125 0.054 (One-Tailed) Delta Scores* GAD 10 μg/ml GAD 2 μg/ml GAD 0.4μg/ml Pair # At Risk Control At Risk Control At Risk Control 1 579 0 5160 768 0 2 3263 0 190 0 199 0 3 0 77 0 5 0 25 4 0 0 10 0 0 0 5 1275 20394 120 53 30 6 1679 992 220 216 77 195 7 2313 1365 0 0 0 70 8 0 0 0 0 00 9 1251 337 0 0 255 21 10 65 391 0 1441 0 0 Mean 1042.4 318.1 133.0178.2 135.3 34.1 Std. Error 357.1 153.0 60.5 142.2 76.0 19.2 WilcoxonP-Value (One-Tailed) 0.018 0.199 0.199

[0074]

1 8 13 amino acids amino acid not relevant linear protein 1 Phe Phe TyrThr Pro Lys Thr Arg Arg Glu Ala Glu Asp 1 5 10 13 amino acids amino acidnot relevant linear protein 2 Phe Trp Tyr Ile Pro Pro Ser Leu Arg ThrLeu Glu Asp 1 5 10 13 amino acids amino acid not relevant linear protein3 Phe Phe Tyr Thr Pro Lys Ser Arg Arg Glu Val Glu Asp 1 5 10 13 aminoacids amino acid not relevant linear protein 4 Phe Phe Tyr Thr Pro MetSer Arg Arg Glu Val Glu Asp 1 5 10 13 amino acids amino acid notrelevant linear protein 5 Phe Trp Phe Val Pro Pro Ser Leu Arg Thr LeuGlu Asp 1 5 10 13 amino acids amino acid not relevant linear protein 6Phe Trp Tyr Ile Pro Gln Ser Leu Arg Gly Val Pro Asp 1 5 10 13 aminoacids amino acid not relevant linear protein 7 Phe Trp Tyr Ile Pro GlnSer Leu Arg Gly Val Pro Asp 1 5 10 16 amino acids amino acid notrelevant linear protein 8 Cys Arg Phe Asp Pro Gln Phe Ala Leu Thr AsnIle Ala Val Arg Lys 1 5 10 15

1. A recombinant or synthetic peptide or chemical equivalent thereofcomprising the formula: X₁X₂X₃ Wherein: X₁ and X₃ may be the same ordifferent and each is an amino acid sequence comprising from 0 to 40naturally or non-naturally occurring amino acid residues; X₂ is anyamino acid sequence of from 10 to 100 residues derived from, homologousto or contiguous within amino acids 506 to 518 inclusive or derivativesthereof of human GAD65 and/or amino acids 24 to 36 inclusive orderivatives thereof of human proinsulin; and wherein said peptidemolecule is capable of reacting with T cells and modifying T-cellfunction when incubated with cells from subjects with pre-clinical orclinical Insulin-Dependent Diabetes Mellitus (IDDM).
 2. A peptidemolecule according to claim 1 wherein X₂ comprises from 10 to 50 aminoacid residues.
 3. A peptide molecule according to claim 2 wherein X₂comprises from 10 to 30 amino acid residues.
 4. A peptide moleculeaccording to claim 3 wherein X₂ comprises from 10 to 15 amino acidresidues.
 5. A peptide molecule according to claim 1 or 2 or 3 or 4wherein X₂ comprises the amino acid sequence: FFYTPKTRREAED.
 6. Apeptide molecule according to claim 1 or 2 or 3 or 4 wherein X₂comprises the amino acid sequence: FWYIPPSLRTLED.
 7. A recombinant orsynthetic peptide or chemical equivalent thereof comprising thesequence: X₁X₂X₃ Wherein: X₁ and X₂ may be the same or different andeach is an amino acid sequence comprising from 0 to 15 naturally ornon-naturally occurring amino acid residues; X₂ is selected fromFFYTPKTRREAED and FWYIPPSLRTLED or a derivative or chemical equivalentthereof and wherein said peptide is capable of reacting with T cells andmodifying T-cell function when incubated with cells from subjects havingpre-clinical or clinical IDDM.
 8. A method of assaying the reactivity ofa subject to IDDM autoantigen said method comprising contacting apeptide or chemical equivalent thereof comprising the formula: X₁X₂X₃Wherein: X₁ and X₃ may be the same or different and each is an aminoacid sequence comprising from 0 to 40 naturally or non-naturallyoccurring amino acid residues; X₂ is any amino acid sequence of from 10to 100 residues derived from, homologous to or contiguous within aminoacids 506 to 518 inclusive or derivatives thereof of human GAD65 and/oramino acids 24 to 36 inclusive or derivatives thereof of humanproinsulin; and wherein said peptide molecule is capable of reactingwith T cells and modifying T-cell function when incubated with cellsfrom subjects having pre-clinical or clinical Insulin-Dependent DiabetesMellitus (IDDM) with cells from said subject and determining reactivityby an appropriate assay.
 9. A method according to claim 8 wherein thecells are selected from the group comprising PBMCs, anti-coagulatedwhole blood and/or tissue biopsy cells.
 10. A method according to claim8 or 9 wherein an appropriate assay includes proliferation assay,cytotoxic assays, celular reactivity or combination thereof.
 11. Amethod according to claim 8 wherein X₂ comprises from 10 to 50 aminoacid residues.
 12. A method according to claim 11 wherein X₂ comprises.from 10 to 30 amino acid residues.
 13. A method according to claim 12wherein X₂ comprises from 10 to 15 amino acid residues.
 14. A methodaccording to claim 8 or 9 or 10 or 11 or 12 wherein X₂ comprises theamino acid sequence: FFYTPKTRREAED.
 15. A method according to claim 8 or9 or 10 or 11 or 12 wherein X₂ comprises the amino acid sequence:FWYIPPSLRTLED.
 16. A method of assaying the reactivity of a subject toIDDM autoantigen said method comprising contacting a peptide or chemicalequivalent thereof comprising the formula: X₁X₂X₃ Wherein: X₁ and X₂ maybe the same or different and each is an amino acid sequence comprisingfrom 0 to 15 naturally or non-naturally occurring amino acid residues;X₂ is selected from FFYTPKTRREAED and FWYIPPSLRTLED or a derivative orchemical equivalent thereof and wherein said peptide is capable ofreacting with T cells and modifying T-cell function when incubated withcells from subjects with pre-clinical or clinical IDDM with cells fromsaid subject and determining reactivity by an appropriate assay.
 17. Amethod according to claim 16 wherein the cells are selected from thegroup comprising PBMCs, anti-coagulated whole blood and/or tissue biopsycells.
 18. A method according to claim 16 or 17 wherein an appropriateassay includes proliferation assay, cytotoxic assays, celular reactivityor combination thereof.
 19. Use of a peptide or chemical equivalentthereof comprising the formula: X₁X₂X₃ Wherein: X₁ and X₃ may be thesame or different and each is an amino acid sequence comprising from 0to 40 naturally or non-naturally occurring amino acid residues; X₂ isany amino acid sequence of from 10 to 100 residues derived from,homologous to or contiguous within amino acids 506 to 518 inclusive orderivatives thereof of human GAD65 and/or amino acids 24 to 36 inclusiveor derivatives thereof of human proinsulin; and wherein said peptidemolecule is capable of reacting with T cells and modifying T-cellfunction when incubated with cells from subjects having pre-clinical orclinical Insulin-Dependent Diabetes Mellitus (IDDM) to assay reactivityof a subject to IDDM autoantigen by contacting said peptide or itschemical equivalent to cells from said subject and determiningreactivity by an appropriate assay.
 20. Use according to claim 19wherein the cells are selected from the group comprising PBMCs,anti-coagulated whole blood and/or tissue biopsy cells.
 21. Useaccording to claim 19 or 20 wherein an appropriate assay includesproliferation assay, cytotoxic assays, cellular reactivity orcombination thereof
 22. Use according to claim 19 wherein X₂ comprisesfrom 10 to 50 amino acid residues.
 23. Use according to claim 22 whereinX₂ comprises from 10 to 30 amino acid residues.
 24. Use according toclaim 23 wherein X₂ comprises from 10 to 15 amino acid residues.
 25. Useaccording to claim 19 or 20 or 21 or 22 or 23 or 24 wherein X₂ comprisesthe amino acid sequence: FFYTPKTRREAED.
 26. Use according to claim 19 or20 or 21 or 22 or 23 or 24 wherein X₂ comprises the amino acid sequence:FWYIPPSLRTLED.
 27. Use of a peptide or chemical equivalent thereofcomprising the formula: X₁X₂X₃ Wherein: X₁ and X₂ may be the same ordifferent and each is an amino acid sequence comprising from 0 to 15naturally or non-naturally occurring amino acid residues; X₂ is selectedfrom FFYTPKTRREAED and FWYIPPSLRTLED or a derivative or chemicalequivalent thereof and wherein said peptide is capable of reacting withT-cells and modifying T-cell function when incubated with cells fromsubjects with pre-clinical or clinical IDDM to assay reactivity of asubject to IDDM autoantigen by contacting said peptide or its chemicalequivalent with cells from said subject and determining reactivity by aproliferation assay.
 28. Use of a peptide or chemical equivalentaccording to claim 27 wherein the cells are selected from the groupcomprising PBMCs, anti-coagulated whole blood and/or tissue biopsycells.
 29. Use of a peptide or chemical equivalent according to claim 27or 28 wherein an appropriate assay includes proliferation assay,cytotoxic assays, celular reactivity or combination thereof.
 30. Amethod of treatment comprising administering to a subject an effectiveamount of a peptide or chemical equivalent thereof for a time and underconditions sufficient to remove or substantially reduce the presence insaid subject of autoreactive T-cells and/or autoantibodies to IDDMautoantigens wherein the peptide comprises the formula: X₁X₂X₃ Wherein:X₁ and X₃ may be the same or different and each is an amino acidsequence comprising from 0 to 40 naturally or non-naturally occurringamino acid residues; X₂ is any amino acid sequence of from 10 to 100residues derived from, homologous to or contiguous within amino acids506 to 518 inclusive or derivatives thereof of human GAD65 and/or aminoacids 24 to 36 inclusive or derivatives thereof of human proinsulin; andwherein said peptide molecule is capable of reacting or modifying T-cellfunction when incubated with cells from subjects having pre-clinical orclinical Insulin-Dependent Diabetes Mellitus (IDDM).
 31. A methodaccording to claim 30 wherein X₂ comprises from 10 to 50 amino acidresidues.
 32. A method according to claim 31 wherein X₂ comprises from10 to 30 amino acid residues.
 33. A method according to claim 32 whereinX₂ comprises from 10 to 15 amino acid residues.
 34. A method accordingto claim 30 or 31 or 32 or 33 wherein X₂ comprises the amino acidsequence: FFYTPKTRREAED.
 35. A method according to claim 30 or 31 or 32or 33 wherein X₂ comprises the amino acid sequence: FWYIPPSLRTLED.
 36. Apharmaceutical composition comprising a recombinant peptide orequivalent thereof according to claim 1 or 7 and one or morepharmaceutically acceptable carriers and/or diluents.